1887

Abstract

Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn reduces tetracycline resistance [ Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). , 20330–20339 ]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short -helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

Keyword(s): TM, transmembrane
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27997-0
2005-07-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/7/mic1512315.html?itemId=/content/journal/micro/10.1099/mic.0.27997-0&mimeType=html&fmt=ahah

References

  1. Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R. & Iwata, S. ( 2003; ). Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615.[CrossRef]
    [Google Scholar]
  2. Aldema, M. L., McMurry, L. M., Walmsley, A. R. & Levy, S. B. ( 1996; ). Purification of the Tn10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein. Mol Microbiol 19, 187–195.[CrossRef]
    [Google Scholar]
  3. Barza, M., Brown, R. B., Shanks, C., Gamble, C. & Weinstein, L. ( 1975; ). Relation between lipophilicity and pharmacological behavior of minocycline, doxycycline, tetracycline, and oxytetracycline in dogs. Antimicrob Agents Chemother 8, 713–720.[CrossRef]
    [Google Scholar]
  4. Brodersen, D. E., Clemons, W. M., Jr, Carter, A. P., Morgan-Warren, R. J., Wimberly, B. T. & Ramakrishnan, V. ( 2000; ). The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103, 1143–1154.[CrossRef]
    [Google Scholar]
  5. Chopra, I. & Roberts, M. ( 2001; ). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65, 232–260.[CrossRef]
    [Google Scholar]
  6. Curiale, M. S., McMurry, L. M. & Levy, S. B. ( 1984; ). Intracistronic complementation of the tetracycline resistance membrane protein of Tn10. J Bacteriol 157, 211–217.
    [Google Scholar]
  7. Deng, W. P. & Nickoloff, J. A. ( 1992; ). Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200, 81–88.[CrossRef]
    [Google Scholar]
  8. Eckert, B. & Beck, C. F. ( 1989; ). Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli. J Biol Chem 264, 11663–11670.
    [Google Scholar]
  9. Guay, G. G., Tuckman, M. & Rothstein, D. M. ( 1994; ). Mutations in the tetA(B) gene that cause a change in substrate specificity of the tetracycline efflux pump. Antimicrob Agents Chemother 38, 857–860.[CrossRef]
    [Google Scholar]
  10. Guillaume, G., Ledent, V., Moens, W. & Collard, J. M. ( 2004; ). Phylogeny of efflux-mediated tetracycline resistance genes and related proteins revisited. Microb Drug Resist 10, 11–26.[CrossRef]
    [Google Scholar]
  11. Hickman, R. K. & Levy, S. B. ( 1988; ). Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli. J Bacteriol 170, 1715–1720.
    [Google Scholar]
  12. Huang, Y., Lemieux, M. J., Song, J., Auer, M. & Wang, D. N. ( 2003; ). Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620.[CrossRef]
    [Google Scholar]
  13. Jewell, J. E., Orwick, J., Liu, J. & Miller, K. W. ( 1999; ). Functional importance and local environments of the cysteines in the tetracycline resistance protein encoded by plasmid pBR322. J Bacteriol 181, 1689–1693.
    [Google Scholar]
  14. Jones, D. T. ( 1999; ). Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292, 195–202.[CrossRef]
    [Google Scholar]
  15. Kimura, T., Suzuki, M., Sawai, T. & Yamaguchi, A. ( 1996; ). Determination of a transmembrane segment using cysteine-scanning mutants of transposon Tn10-encoded metal-tetracycline/H+ antiporter. Biochemistry 35, 15896–15899.[CrossRef]
    [Google Scholar]
  16. Kimura, T., Ohnuma, M., Sawai, T. & Yamaguchi, A. ( 1997; ). Membrane topology of the transposon 10-encoded metal-tetracycline/H+ antiporter as studied by site-directed chemical labeling. J Biol Chem 272, 580–585.[CrossRef]
    [Google Scholar]
  17. Kimura-Someya, T., Iwaki, S., Konishi, S., Tamura, N., Kubo, Y. & Yamaguchi, A. ( 2000; ). Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-tetracycline/H+ antiporter. J Biol Chem 275, 18692–18697.[CrossRef]
    [Google Scholar]
  18. Konishi, S., Iwaki, S., Kimura-Someya, T. & Yamaguchi, A. ( 1999; ). Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H+ antiporter. FEBS Lett 461, 315–318.[CrossRef]
    [Google Scholar]
  19. McGuffin, L. J., Bryson, K. & Jones, D. T. ( 2000; ). The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405.[CrossRef]
    [Google Scholar]
  20. McMurry, L. M. & Levy, S. B. ( 2000; ). Tetracycline resistance in gram-positive bacteria. In Gram-Positive Pathogens, pp. 660–677. Edited by V. Fischetti, R. Novick, J. Ferretti, D. Portnoy & J. Rood. Washington DC: American Society for Microbiology.
  21. McMurry, L., Petrucci, R. E., Jr & Levy, S. B. ( 1980; ). Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A 77, 3974–3977.[CrossRef]
    [Google Scholar]
  22. McMurry, L. M., Stephan, M. & Levy, S. B. ( 1992; ). Decreased function of the class B tetracycline efflux protein Tet with mutations at aspartate 15, a putative intramembrane residue. J Bacteriol 174, 6294–6297.
    [Google Scholar]
  23. Moyed, H. S., Nguyen, T. T. & Bertrand, K. P. ( 1983; ). Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression. J Bacteriol 155, 549–556.
    [Google Scholar]
  24. Nakano, Y., Yoshida, Y., Yamashita, Y. & Koga, T. ( 1995; ). Construction of a series of pACYC-derived plasmid vectors. Gene 162, 157–158.[CrossRef]
    [Google Scholar]
  25. Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr ( 1998; ). Major facilitator superfamily. Microbiol Mol Biol Rev 62, 1–34.
    [Google Scholar]
  26. Rubin, R. A. & Levy, S. B. ( 1990; ). Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family. J Bacteriol 172, 2303–2312.
    [Google Scholar]
  27. Rubin, R. A., Levy, S. B., Heinrikson, R. L. & Kezdy, F. J. ( 1990; ). Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins. Gene 87, 7–13.[CrossRef]
    [Google Scholar]
  28. Saier, M. H., Jr, Beatty, J. T., Goffeau, A. & 11 other authors ( 1999; ). The major facilitator superfamily. J Mol Microbiol Biotechnol 1, 257–279.
    [Google Scholar]
  29. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  30. Sapunaric, F. M. & Levy, S. B. ( 2003; ). Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C). J Biol Chem 278, 28588–28592.[CrossRef]
    [Google Scholar]
  31. Saraceni-Richards, C. A. & Levy, S. B. ( 2000a; ). Evidence for interactions between helices 5 and 8 and a role for the interdomain loop in tetracycline resistance mediated by hybrid Tet proteins. J Biol Chem 275, 6101–6106.[CrossRef]
    [Google Scholar]
  32. Saraceni-Richards, C. A. & Levy, S. B. ( 2000b; ). Second-site suppressor mutations of inactivating substitutions at gly247 of the tetracycline efflux protein, Tet(B). J Bacteriol 182, 6514–6516.[CrossRef]
    [Google Scholar]
  33. Seoane, A., Sabbaj, A., McMurry, L. M. & Levy, S. B. ( 1992; ). Multiple antibiotic susceptibility associated with inactivation of the prc gene. J Bacteriol 174, 7844–7847.
    [Google Scholar]
  34. Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. ( 2001; ). Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter. J Biol Chem 276, 20330–20339.[CrossRef]
    [Google Scholar]
  35. Tuckman, M., Petersen, P. J. & Projan, S. J. ( 2000; ). Mutations in the interdomain loop region of the tetA(A) tetracycline resistance gene increase efflux of minocycline and glycylcyclines. Microb Drug Resist 6, 277–282.[CrossRef]
    [Google Scholar]
  36. Wang, W. & Malcom, B. A. ( 1999; ). Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuickChange Site-Directed Mutagenesis. Biotechniques 26, 680–682.
    [Google Scholar]
  37. Weinglass, A. B. & Kaback, H. R. ( 2000; ). The central cytoplasmic loop of the major facilitator superfamily of transport proteins governs efficient membrane insertion. Proc Natl Acad Sci U S A 97, 8938–8943.[CrossRef]
    [Google Scholar]
  38. Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. ( 1989; ). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469–3478.[CrossRef]
    [Google Scholar]
  39. Yamaguchi, A., Adachi, K. & Sawai, T. ( 1990a; ). Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett 265, 17–19.[CrossRef]
    [Google Scholar]
  40. Yamaguchi, A., Udagawa, T. & Sawai, T. ( 1990b; ). Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem 265, 4809–4813.
    [Google Scholar]
  41. Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T. & Sawai, T. ( 1990c; ). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport. J Biol Chem 265, 15525–15530.
    [Google Scholar]
  42. Yamaguchi, A., Iwasaki-Ohba, Y., Ono, N., Kaneko-Ohdera, M. & Sawai, T. ( 1991; ). Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett 282, 415–418.[CrossRef]
    [Google Scholar]
  43. Yamaguchi, A., Someya, Y. & Sawai, T. ( 1992a; ). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J Biol Chem 267, 19155–19162.
    [Google Scholar]
  44. Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M. & Sawai, T. ( 1992b; ). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices. J Biol Chem 267, 7490–7498.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27997-0
Loading
/content/journal/micro/10.1099/mic.0.27997-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error